The recalcitrance of biofilm communities to a wide range of antimicrobial treatments, including those involving antiseptics, disinfectants and antibiotics, has been pivotal towards the funding and study of biofilm physiology (Gilbert, et al., Adv. Microb. Physiol. 46:203-256, 2002). Intuitive explanations of recalcitrance, such as reaction diffusion, limitation of access, and phenotypic heterogeneity, fail to fully explain the phenomenon (Gilbert, et al., supra, 2002). Since the introduction of antibiotics in the 1940's microbiologists have observed that antimicrobial treatments often fail to completely eradicate the target microbial population (Bigger, The Lancet ii:497-499, 1994; Lewis, Antimicro. Agents Chemo. 45:999-1007,2001, Spoering, et al., J. Bacterial. 183:6746-6751, 2001; Suyfa, et al., Science 2003; Babalan, et al., Science 305:1622-1625, 2004; Levin, Science 305:1678-1679, 2004). The survivors, termed ‘persisters’ appear to be equally sensitive to treatments, when isolated and re-grown, as were the original progenitor population. This argues against chance mutations affecting susceptibility, and indicates a definite role for phenotypic heterogeneity within bacterial cultures and the temporary expression of a resistant phenotype by a small sub-set of cells.
For many bacterial infections, the presence of persisters does not affect the outcome of treatments (Lewis, supra, 2001). This is because the immune system is able to successfully combat such small numbers of surviving cells (Lewis, supra, 2001). In chronic, deep-seated infections, however, and in those associated with growth of the bacteria as biofilms, the persisters are retained within the residual biofilm matrix and physically protected from phagocytosis (Lewis, supra, 2001). Additionally such cells are nurtured by the concentration of nutrients released from a lysing susceptible population and are able to rapidly re-establish themselves post-treatment (Lewis, supra, 2001; Gilbert, et al., supra, 2002).
An intriguing hypothesis, recently proposed by the group of Kim Lewis (Northeastern University, Boston, USA), suggests that death from antimicrobial treatment relates to an induced autolysis of sub-lethally damaged organisms (apoptosis) (Lewis, Microbiol. Mole. Biol. Rev. 64:503-514, 2000). Thus, regardless of the mechanisms of action of the treatment agent, bacterial death could be related to a singular response by the cells. Invocation of a singular mechanism of cell death enabled singular mechanisms of resistance to be postulated. Previous work had suggested that mutant cell lines defective in hipBA operon, and relE were substantially altered in their expression of the persistence phenomenon (Keren, et al., FEMS Microbiol. Let. 230:13-18, 2004).
relE is a general inhibitor of translation and is associated with slow growing or non-growing cells and over-expression causes a dramatic increase in the incidence of persistence. hipB is a transcriptional regulator and suppresses hipA (Black, et al., J. Bacteriol. 176:4081-4091, 1994). HipA does not show homology to any protein of known function but is postulated to encode a toxin-antitoxin module (Black, et al., J. Bacteriol. 173:5732-5739, 1991). The toxin component is relatively stable whilst the anti-toxin component is labile (Moyed, et al., J. Bacteriol. 166:399-403, 1986; Black, et al., supra, 1994; Falla, et al., Antimicrob. Agents Chemo. 42:3282-3284, 1998). Imbalance of the pair is thought to lead to cell death and subsequent cellular lysis. Thus, Lewis proposed that persistence related to loss, or suppression, of this hypothetical apoptotic mechanism.
Lewis (Keren, et al., supra, 2004) went on to examine the pattern of gene expression in isolated persister cells using DNA expression arrays. Persisters were obtained by treating an exponentially growing culture with a β-lactam antibiotic. Actively growing cells lysed as a result of defects in cell wall synthesis whilst the persisters, reputedly slow growing or quiescent (Sufya, et al., J. App. Microbiol. 95:1261-1267, 2003), did not succumb. Prolonged (180 min) incubation of the culture led to denaturation of that mRNA released from the lysed cells and enabled the persisters to be collected and their RNA to be extracted. Whilst the recovered persisters had undoubtedly tolerated the insult of antibiotic exposure they had also been exposed to ampicillin for a prolonged period and might have exhibited changes related to non-lethal actions of the agent. DNA expression arrays developed with the extracted RNA showed a number of genes and gene products to be up regulated. Several functional groups of genes were evident within the most upregulated 300 genes. These were found to represent the SOS response genes, recA, sulA and uvrBA together with umuDC; the phage shock (psp) operon genes, and several heat and cold shock genes. All of these patterns of gene expression were consistent with the persisters being slow growing or dormant but could not explain the phenomenon of persistence. Other genes were upregulated and represented toxin-antitoxin modules, including dinJlyafQ, yefM, relBE and mazEF, adding support to his proposed mechanisms of persistence (Lewis, supra, 2000; Kaldalu, et al., Antimicrob. Agents Chemo. 48:890-896, 2004; Keren, et al., supra, 2004).